Project Summary/Abstract Human immunodeficiency virus type-1 (HIV-1) is a retrovirus that infects CD4+ T cells of the immune system. If left untreated, HIV-1 infected individuals will progress to AIDS and may ultimately die as a result. Combination antiretroviral therapy is extremely effective at stopping the replication of HIV-1 in infected individuals. Despite the success of this therapy at suppressing HIV-1 replication to clinically undetectable levels, antiretroviral therapy is not curative. This is due to the persistence of HIV-1 in a silent, or latent, state within a subset of CD4+ T cells known as resting memory CD4+ T cells. In this latent state, these infected cells are not targeted by antiretroviral drugs and cannot be eliminated by the immune system. In HIV-1 infected individuals, latently infected CD4+ T cells are found at extremely low frequencies (~1 per million resting memory CD4+ T cells). However, this population of latently infected cells is very stable, demanding that HIV-1 infected individuals remain on antiretroviral therapy indefinitely. Therefore, this population of latently infected CD4+ T cells is the main barrier to curing HIV-1 infection. Developing strategies to eliminate latently infected cells is a major focus of the NIH, NIAID, and the HIV-1 research field. To demonstrate the efficacy of therapeutics targeting the latent reservoir, we must be able to measure the frequency of latently infected cells using rapid and accurate assays that can be scaled for widespread clinical use. However, such assays are not currently available. Accelevir Diagnostics, LLC is therefore developing a new molecular assay to accurately measure the size of the latent reservoir by quantifying the number of intact, replication competent proviruses present in CD4+ T cells from HIV-1 infected patients. Broadly, this proposal aims to define critical assay kit controls, perform analytical validation studies, benchmark our novel assay against available research assays for latent HIV-1, collect baseline data from HIV-1 infected individuals on suppressive therapy, produce commercial prototype kits for beta testing, and develop a data analysis framework for assay readouts and associated clinical metadata. The overall goals of this proposal are to complete development of our commercial test for latent HIV-1 and collect the foundational performance data needed to enable market entry. !